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Development of a thiol-ene based screening platform for enzyme immobilizationdemonstrated using horseradish peroxidase

Hoffmann, Christian; Pinelo, Manuel; Woodley, John; Daugaard, Anders Egede

Published in:Biotechnology Progress

Link to article, DOI:10.1002/btpr.2526

Publication date:2017

Document VersionPeer reviewed version

Link back to DTU Orbit

Citation (APA):Hoffmann, C., Pinelo, M., Woodley, J., & Daugaard, A. E. (2017). Development of a thiol-ene based screeningplatform for enzyme immobilization demonstrated using horseradish peroxidase. Biotechnology Progress, 33(5),1267-1277. https://doi.org/10.1002/btpr.2526

https://doi.org/10.1002/btpr.2526

https://orbit.dtu.dk/en/publications/e09e06ef-6c8c-4d92-9753-bb14316c3ef3

https://doi.org/10.1002/btpr.2526

Development of a thiol-ene based screening platform for enzyme immobilization

demonstrated using horseradish peroxidase

Christian Hoffmann, Manuel Pinelo, John M. Woodley, Anders E. Daugaard*

C. Hoffmann, Assoc. Prof. A. E. Daugaard

Danish Polymer Centre, Department of Chemical and Biochemical Engineering, Technical

University of Denmark, Søltofts Plads Building 229, 2800 Kgs. Lyngby, Denmark

*Corresponding author: [email protected]

Assoc. Prof. M. Pinelo

Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering,

Technical University of Denmark, Søltofts Plads Building 229, 2800 Kgs. Lyngby, Denmark

Prof. J. M. Woodley

Process and Systems Engineering Center (PROSYS), Department of Chemical and

Biochemical Engineering, Technical University of Denmark, Søltofts Plads Building 229,

2800 Kgs. Lyngby, Denmark

Keywords: thiol-ene chemistry, surface functionalization, enzyme immobilization, enzyme-

surface interaction

Biocatalysts and Bioreactor Design Biotechnology ProgressDOI 10.1002/btpr.2526

This article has been accepted for publication and undergone full peer review but has not beenthrough the copyediting, typesetting, pagination and proofreading process which may lead todifferences between this version and the Version of Record. Please cite this article asdoi: 10.1002/btpr.2526© 2017 American Institute of Chemical Engineers Biotechnol ProgReceived: Apr 25, 2017; Revised: Jul 07, 2017; Accepted: Jul 10, 2017

This article is protected by copyright. All rights reserved.

http://orcid.org/0000-0002-0627-6310

2

Abstract

Efficient immobilization of enzymes on support surfaces requires an exact match between the

surface chemistry and the specific enzyme. A successful match would normally be identified

through time consuming screening of conventional resins in multiple experiments testing

individual immobilization strategies. In this study we present a versatile strategy that largely

expands the number of possible surface functionalities for enzyme immobilization in a single,

generic platform. The combination of many individual surface chemistries and thus

immobilization methods in one modular system permits faster and more efficient screening,

which we believe will result in a higher chance of discovery of optimal surface/enzyme

interactions.

The proposed system consists of a thiol-functional microplate prepared through fast

photochemical curing of an off-stoichiometric thiol-ene (OSTE) mixture. Surface

functionalization by thiol-ene chemistry (TEC) resulted in the formation of a functional

monolayer in each well, whereas, polymer surface grafts were introduced through surface

chain transfer free radical polymerization (SCT-FRP). Enzyme immobilization on the

modified surfaces was evaluated by using a rhodamine labeled horseradish peroxidase (Rho-

HRP) as a model enzyme, and the amount of immobilized enzyme was qualitatively assessed

by fluorescence intensity (FI) measurements. Subsequently, Rho-HRP activity was measured

directly on the surface. The broad range of utilized surface chemistries permits direct

correlation of enzymatic activity to the surface functionality and improves the determination

of promising enzyme-surface candidates. The results underline the high potential of this

system as a screening platform for synergistic immobilization of enzymes onto thiol-ene

polymer surfaces.

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1. Introduction

Immobilized enzymes are widely used as biosensors, in drug delivery, as well as in dairy and

food processes for the production of pharmaceuticals and cosmetics.1,2 Their high substrate

specificity, selectivity, and efficiency at mild reaction conditions, such as ambient

temperature, physiological pH and atmospheric pressure make them interesting alternatives to

conventional catalysts. However, as functional biocatalysts, enzymes frequently demonstrate

limited operational stability. Immobilization of enzymes has generally been demonstrated to

have a stabilizing effect due to stronger confinement in enzyme rigidity, which leads to

improved pH, organic solvent and thermal stability.3–5 However, increased stability and even

improved selectivity or activity can only be achieved, when a favorable environment for the

enzyme is obtained. Further advantages are the improved recovery and purification from

reaction media and potential recirculation of immobilized biocatalysts in the process.6

Depending on the application and the particular enzyme of interest, many strategies are

available for immobilization with, or without, a support matrix.7,8 Cross-linking enzymes by

either physical or covalent interactions, leads to the formation of enzyme aggregates, which

can directly be used.9 Entrapment or binding to a support matrix requires the use of a solid

carrier material.10 However, a general concern of most of these procedures is the potential

reduction in biocatalytic activity upon immobilization due to a combination of various

individual factors, such as changes in conformation and accessibility to the active site.11 These

factors are greatly influenced when confining structural freedom or even changing their

structural conformation upon immobilization.12 In some specific cases, strong attachment of

enzymes to a surface, for instance by multibond attachment, might also have a beneficial

effect for the activity in addition to improvements in stability due to conformational

restrictions of the enzyme.13

For the attachment of enzymes to the surface of solid supports various factors for stabilization

and destabilization have been identified and reviewed by Talbert and Goddard.14 In order to

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prevent deactivation, the properties of the surface (hydrophilicity, hydrophobicity, charge,

surface topology, and functionality) have to match structural and compositional characteristics

of a particular enzyme and provide a favorable local environment for immobilized enzyme.

15 Each enzyme or protein has a unique structure and generalizations about stabilization

effects on specific surfaces are therefore not universal applicable. For this reason different

approaches have been investigated in order to achieve surface modifications, which can lead

to improved biocatalytic activity of immobilized enzymes. Surface functionalizations with

charged16,17, hydrophilic18 or hydrophobic19 moieties as well as functional groups, to which

enzymes can bind covalently20, have been investigated. Direct attachment to the surface via

covalent binding leads to structurally confined and rigid immobilized enzymes with strong

interactions between the surface and the enzyme.14 Polymers grafted to or from a surface21–23

as well as surface bound tethers24,25 can act as spacers between surface and enzyme, which

offers higher mobility of the enzyme. This may either result in an improved enzyme activity,

or this may negatively impact the stability due to higher mobility and reduced rigidity.

Consequently, the final result on activity and stability will be a balance between several

effects, which should be identified for each specific enzyme.

Different materials like glass, metals or polymers can serve as a solid support for enzyme

immobilization. The easy processibility of polymers, adjustable mechanical properties,

conductivity, and ease of post-functionalization make polymers an extensively used support

material.26 Recently, the development of stoichiometric thiol-ene (STE) and furthermore off-

stoichiometric thiol-ene (OSTE) polymer thermosets with tunable mechanical properties was

reported.27–30 OSTE materials show high compatibility with many solvents, are thermally

stable and can directly be surface functionalized after preparation. Here, thiol and alkene

containing monomers undergo cross-linking by thiol-ene chemistry (TEC) under radical

conditions. TEC is an efficient method providing high yields under relatively mild reaction

conditions.31 This highly modular system has allowed STE and OSTE materials to be applied

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for fabrication of microfluidics,32,33 particles34, hydrogels35 or high internal phase emulsions

(HIPE).36 Variations in stoichiometry between the reagents lead to either unreacted thiol or

alkene groups, which are present in the bulk OSTE material as well as on the surface. This

excess of functional groups can be used further for surface modification via TEC.37–40 Hence,

surface hydrophilization, hydrophobization, as well as introduction of biological moieties has

been reported.41–43 Post preparation modification of the materials has even been shown to be

possible by incorporation of glycidyl methacrylate into a photocurable TE material, which

resulted in unreacted epoxide groups after curing. These residual functionalities were used for

covalent immobilization of α-amylase for the preparation of a biocatalytic surface.44

However, due to the vast amount of existing enzyme support resins, the selection of a suitable

candidate for specific enzymes is very difficult, as a beneficial match surface-enzyme cannot

be generally predicted by just considering the chemistry of the support surface and its

potential interaction with the enzyme. For instance, these interactions can be of hydrophilic or

hydrophobic nature as well as formed hydrogen bonds, which show a different influence on

different enzymes and thus, impact their biocatalytic performance. Therefore, the objective of

this study was to develop a single, versatile platform that allows broader screening of different

surface chemistries for enzyme immobilization. For this purpose the high modularity in

preparation of TE materials and their possibility for facile surface modification was exploited

to illustrate their potential as support for the immobilization of enzymes. An STE/OSTE

microplate was fabricated, which enabled versatile surface functionalization via

photochemical TEC or in a new approach through surface chain transfer free radical

polymerization (SCT-FRP). Initial activity measurements of immobilized horseradish

peroxidase as a model enzyme on those surfaces were conducted, which were used for

evaluation of beneficial surface chemistries as enzyme support. Such a microplate permits

screening of synergistic interactions between enzymes and surfaces in a time-saving,

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systematic approach, which should enable facile identification of efficient immobilization

systems.

2. Materials and Methods

Materials

Pentaerythritol tetrakis(3-mercaptopropionate) (PETMP, >95%), 1,3,5-Triallyl-1,3,5-triazine-

2,4,6(1H,3H,5H)-trione (TATATO, 98%), allyl pentafluorobenzene (APFB, >99%),

allyltrifluoroacetate (ATFA , 98%), allyl alcohol (AA, 99%), allyl malonic acid (AMA,

≥98%), 1-vinyl imidazole (Vim, ≥99%), allylamine hydrochloride (Aam, 98%), [2-

(Methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (97%) as a

sulfobetaine methacrylate (SBMA), 2,2-dimethoxy-2-phenylacetophenone (DMPA, 99%),

allyl glycidyl ether (AGE, >99%), horseradish peroxidase (HRP, lyophilized powder, 50-150

U mg-1), 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS,

≥98%) and hydrogen peroxide (H2O2, 3%) were obtained from Sigma Aldrich and used

without further purification.

Methoxy poly(ethylene glycol) monomethacrylate (MPEGMA, Mn 500), glycidyl

methacrylate (GMA, 97%) and 2,2,3,3,4,4,5,5 octafluoropentyl acrylate (OFPA, 97%) were

purchased from Sigma Aldrich and passed through a short plug flow column containing

aluminum oxide (Sigma-Aldrich, activated, basic, Brockmann I, standard grade) prior to use.

Ethanol was obtained from VWR Chemical. Lucirin TPO-L (ethyl-2, 4, 6-tri-

methylbenzoylphenyl phosphinate) was obtained from IGM Resins. Sylgard 184 –

poly(dimethylsiloxane) (PDMS) elastomer kit was purchased from Dow Corning.

Characterization.

Fourier transform infrared (FT-IR) spectroscopy was carried out using a Nicolet iS50 FT-IR

fitted with a diamond crystal attenuated total reflection accessory (ATR), which operated at a

resolution of 4 cm-1 and 32 scans per measurement and was used to identify chemical

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modifications made by surface functionalization. XPS experiments were conducted on a

Thermo Fisher Scientific K-Alpha (East Grinstead, UK). Large area surface analysis used a

400 µm spot of monochromatized aluminum Kα radiation, following which survey (pass

energy 200 eV) and high-resolution (pass energy 50 eV) spectra for relevant elements were

acquired. Data analyses of the obtained XPS spectra were performed using the Avantage

software package as provided by the manufacturer. Average values and standard deviations

were conducted for each surface in technical replicates of three (n=3). Static water contact

angles (WCAs) of the virgin and functionalized OSTE surfaces were determined by using a

Dataphysics Contact Angle System OCA20. Each surface was tested via the static sessile drop

method at 23°C, the average value and standard deviations were determined from three

technical replicates (n=3).

The immobilization yield of rhodamine labeled HRP in the wells was determined using a

POLARstar® Omega from BMG Labtech equipped with a fluorescence probe (gain of 1000

as a general setting of the instrument) at 25 °C. This value was subtracted by the fluorescence

intensity result measured prior enzyme immobilization. The enzyme activity of immobilized

Rho-HRP was determined by absorbance measurements (at 414 nm) using a POLARstar®

Omega from BMG Labtech equipped with a UV-VIS probe (20 scans per measurement) at 25

°C. For each surface, immobilization of Rho-HRP including FI and enzyme activity

measurements, were performed in experimental replicates of three (n=3), from which the

average values and standard deviations were calculated.

Preparation of OSTE microtiter plate.

In a stoichiometric ratio between thiol and ene groups, PETMP (24.44 g, 0.050 mol, 0.20 mol

thiol), TATATO (16.64 g, 0.067 mol, 0.20 mol ene) and TPO-L (11.1 mg, 0.03 wt%) were

mixed with a dual asymmetric centrifuge Speed-Mixer, High Wycombe, UK, DAC 150 FVZ-

K at 2500 to 3500 r.p.m. for 2 min screened from ambient light. A previously prepared PDMS

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mold with the negative imprint of the top part of the microtiter plate was filled with this

mixture, which was subsequently cured under UV light (λ= 365 nm, 2.9 mW cm-2) for 3 min.

Then, PETMP (22.06 g, 0.045 mol, 0.18 mol thiol), TATATO (7.91 g, 0.032 mol, 0.095 mol

ene) and TPO-L (8.1 mg, 0.03 wt%) were mixed in a Speed-mixer and a second PDMS mold

for the bottom component was filled with this mixture. This mixture was left under sunlight

for 7 min and then the previously top component was attached from the top. This assembly

was again cured under UV light (λ= 365 nm, 2.9 mW cm-2) for 3 min resulting in the final

microwell plate. A schematic drawing with measures of the top and bottom component of the

microwell plate separately is shown in Figure S1.

IR (cm-1): 2961 (C-H), 2571 (S-H), 1731 (C=O), 1678 (C=Calkene), 1459 (O-CH2 ester), 1351,

1141 (C-O-Cstretch vibr), 1021 (C-O-C), 764(C-O-Cdeformation), 528.

Surface functionalization via TEC with allyl pentafluorobenzene (OSTE-APFB).

In a general functionalization procedure, an ethanolic solution (10 mL) containing APFB

(0.460 mL, 3.0 mmol) and TPO-L (9.9 mg, 1 mol%) was prepared screened from ambient

light. 300 µL of this solution was added to a single well and subsequently irradiated with UV

light (λ= 365 nm, 2.9 mW cm-2) for 5 min. Subsequently, the well was thoroughly rinsed with

Ethanol and finally blow dried with air. Typically, five replicates of each modification were

prepared.


1140 (C-O-Cstretch vibr), 1022 (C-O-C), 764 (C-O-Cdeformation), 528.

Preparation of OSTE-ATFA.

OSTE-ATFA was prepared in accordance with the general procedure, using 300 µL of an

ethanolic solution (10 mL) of allyltrifluoroacetate (ATFA, 0.27 mL, 2.1 mmol) and TPO-L

(9.0 mg, 0.7 mol%) as reagents.

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Preparation of OSTE-OFPA. OSTE-OPFA was prepared in accordance with the general

procedure, using 300 µL an ethanolic solution (10 mL) of 2,2,3,3,4,4,5,5 octafluoropentyl

acrylate (OFPA, 0.58 mL, 3.0 mmol) and TPO-L (9.3 mg, 0.97 mol%) as reagent.



Preparation of OSTE-AA. OSTE-AA was prepared in accordance with the general procedure,

using 300 µL of an ethanolic solution (10 mL) of allyl alcohol (AA, 0.20 mL, 3.0 mmol) and

TPO-L (10.5 mg, 1.1 mol%) as reagent.



Preparation of OSTE-AMA. OSTE-AMA was prepared in accordance with the general

procedure, using 300 µL of an ethanolic solution (10 mL) of allyl malonic acid (AMA, 0.433

g, 3.0 mmol) and TPO-L (10.4 mg, 1.1 mol%) as reagent.



Preparation of OSTE-Vim. OSTE-Vim was prepared in accordance with the general

procedure, using 300 µL of an ethanolic solution (10 mL) of 1-vinyl imidazole (Vim, 0.30

mL, 3.0 mmol) and TPO-L (9.2 mg, 1.0 mol%) as reagent.

IR (cm-1): 3136 (N-H), 2961 (C-H), 2571 (S-H), 1730 (C=O), 1677 (C=Calkene), 1459 (O-CH2

ester), 1352, 1139 (C-O-Cstretch vibr), 1024 (C-O-C), 763 (C-O-Cdeformation), 528.

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Preparation of OSTE-AGE. OSTE-AGE was prepared in accordance with the general

procedure, using 300 µL of an ethanolic solution (10 mL) of allyl glycidyl ether (AGE, 0.36

mL, 3.0 mmol) and TPO-L (9.0 mg, 0.97 mol%) as reagent.



Preparation of OSTE-Aam hc. OSTE-Aam hc was prepared in accordance with the general

procedure, using 300 µL of an ethanolic solution (10 mL) of allylamine hydrochloride (Aam

hc, 0.281 g, 3.0 mmol) and TPO-L (10.1 mg, 1.1 mol%) as reagent.



Surface functionalization via surface chain transfer free radical polymerization (SCT FRP)

with poly(ethylene glycol) methylether methacrylate (OSTE-g-pMPEGMA).

In a general procedure Methoxy poly(ethylene glycol) monomethacrylate (MPEGMA, 0.92

mL, 2.0 mmol) and 2,2-dimethoxy-2-phenylacetophenone (DMPA, 2.6 mg, 0.50 mol%) were

dissolved in ethanol (1.85 mL) screened from ambient light. 300 µL of this solution was

added to a single well and subsequently irradiated with UV light (λ= 365 nm, 2.9 mW cm-2)

for 30 min. Subsequently, the well was thoroughly rinsed with Ethanol and finally blow dried

with air. Typically, five wells in the same well plates were functionalized.

IR (cm-1): 2871 (C-H), 1733 (C=O), 1684 (C=Calkene), 1461 (O-CH2 ester), 1349, 1101 (C-O-

Cstretch vibr), 1035 (C-O-C), 944, 852, 764 (C-O-Cdeformation), 528.

Preparation of OSTE-g-pGMA. OSTE-g-pGMA was prepared in accordance with the general

procedure, using 300 µL of a solution of glycidyl methacrylate (GMA, 0.453 mL, 3.3 mmol),

DMPA (4.3 mg, 0.5 mol%) in ethanol (0.91 mL) as reagents.

IR (cm-1): 2999 (C-Hepoxide), 2936 (C-H), 1724 (C=O), 1685 (C=Calkene), 1449 (O-CH2 ester),

1129 (C-Ostretch vibr), 989 (C-O), 904 (epoxide ring vibr), 841 (C-C), 757 (C-O-Cdeformation).

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Preparation of OSTE-g-pSBMA. OSTE-g-pSBMA was prepared in accordance with the

general procedure, using 300 µL of a solution of [2-(methacryloyloxy)ethyl]dimethyl-(3-

sulfopropyl)ammonium hydroxide (SBMA, 0.846 g, 3.0 mmol), DMPA (3.9 mg, 0.5 mol%)

in ethanol (1.28 mL) as reagents.

IR (cm-1): 3443 (N-H), 2964 (C-H), 1726 (C=O), 1676 (C=Calkene), 1459 (O-CH2 ester), 1143

(C-Ostretch vibr), 1035, 901, 762 (C-O-Cdeformation), 603, 524.

Preparation of OSTE-g-pOFPA. OSTE-g-pOFPA was prepared in accordance with the

general procedure, using 300 µL of a solution of 2,2,3,3,4,4,5,5 octafluoropentyl acrylate

(OFPA, 1.25 mL, 3.0 mmol), DMPA (3.1 mg, 0.4 mol%) in ethanol (0.93 mL) as reagents.

IR (cm-1): 2968 (C-H), 1737 (C=O), 1684 (C=Calkene), 1464 (O-CH2 ester), 1163 (C-F), 1124

(C-Ostretch vibr), 1044, 805, 765 (C-O-Cdeformation), 539 (C-F).

Labelling of horseradish peroxidase.

Horseradish peroxidase (10.0 mg) was dissolved in sodium carbonate buffer (0.1M, pH 9.5,

1.0 mL) and 20 µL of sulforhodamine B acid chloride in DMF (10 mg mL-1) was added

dropwise under mild vortex mixing was added. This solution was incubated at 4 °C overnight

and subsequently purified by dialysis for 3 days against PBS buffer. The purified solution was

stored at 4 °C for further use.

Immobilization of horseradish peroxidase.

The previously labelled Rho-HRP solution (0.455 mL) was diluted with PBS buffer (pH 7.3,

1.0 mL), and 300 µL of this solution was added to each well. In order to prevent evaporation

of liquid the plate was covered with a PDMS cover and incubated at 4 °C for 16 h. Then, the

supernatant was removed and PBS buffer (300µL) was added to the well. The rinsing buffer

was replaced 4 times while slowly shaking. The immobilization yield was determined via

fluorescence intensity measurement of the surface.

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Activity assay of bioactive surfaces.

A phosphate buffer solution (0.1M, pH5, 300 µL) containing 2,2′-azino-bis(3-

ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS, 1.0 mM) and hydrogen

peroxide (10 mM) was added into a well with immobilized Rho-HRP. In the presence of H2O2,

ABTS (absorbance maximum at 340 nm) is oxidized by HRP to ABTS●+, which shifts the

absorbance maximum to 412 nm (ε412=3.6 x 104M-1 cm-1).45

The absorbance at 412 nm corresponding to the absorbance maximum of the oxidized product

(ABTS●+) was measured, which corresponds directly to the concentration of the product. The

measurements were conducted every 20 s over 10 min directly through the well including

shaking (10s, 300 r.p.m.) between each measurement. The slope of the measurements was

used as a measure for the activity of the immobilized Rho-HRP on the surface.

3. Results and Discussion

The preparation of STE/OSTE microplates was based on a 2-step curing process via

photoinitiated TEC using pentaerythritol tetrakis(3-mercaptopropionate) (PETMP) and 1,3,5-

triallyl-1,3,5-triazine-2,4,6(1H,3H,5H)-trione (TATATO) as shown in Figure 1A.

PDMS mold

STE

cured STE

OSTE (excess 90% thiol)PDMS mold

cured OSTE (90%thiol)

UV light (365 nm)

UV light (365 nm)

1

2

3

SH SH SH

A B

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Figure 1 (A) schematic representation of the microplate preparation using a 2-step curing

process, (1) curing by irradiation with UV light of the top part prepared from a stoichiometric

thiol-ene (STE) mixture of PETMP and TATATO in a PDMS mold, (2) the prepared top part

is placed on a OSTE mixture (excess 90% thiol) of PETMP and TATATO, where a second

curing step by irradiation with UV light leads to the final microplate, (3) which contain excess

thiol groups in the bottom and STE on the well walls, (B) Photographs of the final microwell

plate.

In a first stage, the top part of the microplate in a STE mixture of PETMP and TATATO was

photochemically cured in the presence of TPO-L in a poly(dimethylsiloxane) (PDMS) mold,

as shown in Figure 1A-1. The mold consisted of the outer geometries of the microplate (127.7

x 85.6 mm2), pillars with the size of the round wells (diameter 15.1 mm) and a depth of 5 mm.

A second mold with a depth of 1.5 mm and the outer diameters of the microtiter plate was

then applied for the second curing step. Here, an OSTE composition of PETMP, TATATO

and TPO-L using a 90 % excess of thiol was applied for the well bottom. The previously

prepared STE top part was then placed on top of the uncured OSTE mixture and both parts

were cured together (see Figure 1A-2). This preparation process resulted in a fully sealed

microplate consisting of 24 wells with a depth of 5 mm, shown in Figure 1A-3 and Figure 1B.

Due to the used compositions, excess thiol groups remained on the bottom of each well. The

side walls of the wells, based on a STE composition, did not contain any residual thiols, as

determined using Ellman’s reagent, which is commonly used for quantification of thiol groups

either on surfaces or in solution (see Figure S2).46 From these results it could be seen that

Ellman’s reagent, which was added to a STE surface, exhibit a very low absorbance compared

to the OSTE with 90 % excess of thiols. Therefore, it was deduced that the number of thiols

on the side walls was considered negligible and that modification of surface bound thiols

occurred exclusively on the bottom surfaces. Furthermore, the transmittance of the OSTE

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material was measured in order to validate the possible application in colorimetric assays. The

material demonstrated a strong absorbance in the UV range from 220 to 340 nm, as shown in

Figure S3. Between 340 and 410 nm, the transmittance increased up to 36 % (absorbance =

0.44) and at any wavelength above, the material was completely transparent (absorbance <

0.15). In general, the absorbance of the STE/OSTE microplate does not substantially differ

from a commercial polystyrene microplate, as indicated in Figure S3. Therefore, the

STE/OSTE plate is well suited for any application in which absorbance measurements of

solutions or surface modifications are performed.

Excess thiol groups on the bottom surface originating from the OSTE mixture allow

controlled surface functionalization via TEC and SCT-FRP. In this study, the surface

modification using both methods has been attempted with a large range of monomers in order

to introduce different types of functionalities as illustrated in Scheme 1.

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Scheme 1. Surface functionalization of thiols from OSTE microwell surfaces via two

different routes; left: surface thiol-ene chemistry (TEC) with allyl alcohol (AA), allyl malonic

acid (AMA), allyl pentafluorobenzene (APFB), allyl trifluoroacetate (ATFA), 2,2,3,3,4,4,5,5

octafluoropentyl acrylate (OFPA), allyl amine hydrochloride (Aam hc), 1-vinyl imidazole

(Vim) and allyl glycidyl ether (AGE) at low concentration (0.3 M) leading to a functional

monolayer on the surface; right: SCT-FRP with methoxy poly(ethylene glycol) methacrylate

(MPEGMA), zwitterionic sulfobetaine methacrylate (SBMA, via [2-

(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide), OFPA and glycidyl

methacrylate (GMA) based monomers at high concentration (0.7 – 2.4 M), leading to a

functional polymer grafted surface.

S

FF

F

F

F S

OH

S

OH

O

HO

O

S

NH2

S

O

O

O

S

AGE

AA

APFB

AMA

Aam hc

VIm

i

MPEGMA

S

O

O F F

F F F F

F

F

S

O

O

O

S

O

O

N SO3

SBMA

GMA

ii

OFPA

S

O

O

F

FF

S

O

O

FF

FF

FF

F

F

O

O

OFPA

ATFA

S

NN

i

i

i

i

i

i

i

ii

ii

ii

Surface thiol-ene chemistry (TEC) Surface chain transfer FRP

SH

OSTE-AAOSTE-AMA

OSTE-APFB

OSTE-ATFA

OSTE-OFPA

OSTE-Aam hc

OSTE-Vim

OSTE-AGE

OSTE-g-pMPEGMA

OSTE-g-pSBMA

OSTE-g-pOFPA

OSTE-g-pGMA

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With the aim to develop a platform for enzyme immobilization, which enables screening of

various surface functionalities and their impact on the activity of surface bound enzymes, a

variety of functional monomers were utilized under TEC conditions. Based on different types

of possible interactions and immobilization mechanisms various reactive moieties were

selected. Hydroxyl functional groups (AA) were introduced as well as fluorine groups (APFB,

ATFA and OFPA). pH responsive modifications, with either acidic groups (AMA) or basic

functionalities, such as amine (Aam hc) and imidazole (Vim) were used as well. This range of

introduced surface chemistries offered possible immobilization mechanisms including

hydrophilic (with OSTE-AA, -AMA), hydrophobic (OSTE-APFB, -ATFA, -OFPA) and ionic

interactions (OSTE-Vim and -AMA). Furthermore, epoxides were introduced (AGE) allowing

bioconjugation through covalent enzymes binding with amine, thiol, imidazole or phenolic

moieties from the enzyme.6 The application of such a broad range of reagents demonstrates

the great versatility of this process and can extensively be expanded, since TEC offers a vast

range of surface chemistries by reaction of any allyl, vinyl or acrylic compound onto the

screening platform. Photochemical surface TEC was performed with low monomer

concentrations (0.3 M) in ethanol solutions in order to prevent polymerization reactions. The

IR spectrum of the starting material (OSTE) contains typical alkane (C-H, 2968 cm-1),

carbonyl (C=O, 1729 cm-1), alkene (C=C, 1683 cm-1) and aliphatic ester (C-O-C, 1141 cm-1)

elements. Comparison with TEC grafted surfaces did not exhibit significant differences, as

illustrated in Figure S4. This could be explained by a low surface coverage due to a TE

addition on the surface. Small structural changes as a result of monolayer formation can

generally not be detected by attenuated total reflectance (ATR) FT-IR due to the domination

of the bulk material in the spectrum, caused by the penetration depth of the IR signal.

However, by using X-ray photoelectron spectroscopy (XPS) in combination with static water

contact angle (WCA) measurements, the individual modifications were confirmed, as shown

in Table 1 for the virgin (OSTE) and TEC modified surfaces.

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Table 1. XPS data and static water contact angles (WCA) of virgin and functionalized OSTE surfaces via TEC with a variety of ene compounds

C1s O1s N1s S2p F1s WCA

[atom%] [atom%] [atom%] [atom%] [atom%] [°]

OSTE 61.1±0.6

(60.6) 23.3±0.2

(24.9) 4.5±0.3

(4.1) 11.1±0.3

(10.4) 67.0±1.2

OSTE-APFB

58.8±2.8 27.2±2.0 2.7±0.0 9.9±0.5 1.4±0.3 62.4±2.1

OSTE-ATFA

55.3±0.0 28.6±0.1 5.2±0.2 8.1±0.2 2.8±0.1 41.1±2.4

OSTE-OPFA

53.4±0.4 26.9±0.1 4.5±0.3 8.1±0.0 7.1±0.6 77.7±4.1

OSTE- AA

60.4±0.2 27.6±0.7 4.5±0.5 7.6±0.4 35.0±1.5

OSTE-AMA

61.3±0.1 27.8±0.1 4.2±0.0 6.7±0.1 25.1±3.6

OSTE- Vim

63.3±1.5 22.6±0.8 7.3±0.2 6.8±0.5 38.5±3.6

OSTE-AGE

58.7±0.6 28.7±0.3 4.2±0.3 8.4±0.2 55.9±3.7

OSTE- Aam hc

59.3±0.2 26.5±0.1 5.5±0.1 8.7±0.1 55.4±4.7

In parenthesis, theoretical atom composition of the virgin surface (OSTE), results are based

on three replicated measurements on the same surface (n=3)

The atom composition of the unreacted OSTE surface estimated by XPS was in agreement

with the theoretical values and therefore used for further comparisons with modified surfaces.

Surface functionalization with fluorinated reagents such as APFB, ATFA and OFPA was

confirmed by the presence of fluorine atoms (F1s) in the XPS spectra, which was not detected

on the native OSTE surface. APFB modified OSTE exhibited with 1.4 atom% the lowest

fluorine content, which suggested a low grafting efficiency. This could be improved by

reaction with ATFA and OFPA (2.8 and 7.1 atom%). In contrast to the allylic reagents, OFPA

being an acrylic monomer is prone to undergo polymerization under radical conditions. Here,

the overall atom composition of OFPA modified surfaces under TEC conditions, showed

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additional to the increase in fluorine content a slight reduction in carbon, sulfur and nitrogen.

This confirmed that under the applied low concentration TEC conditions, even for an acrylic

monomer, like OFPA, the addition reaction dominated, leading to the formation of a

monolayer (polymerization could then be suppressed). The atom composition after surface

TEC with AMA, AA and AGE did not differ significantly from the reference OSTE surface.

This could be attributed to similar theoretical atom composition of these reagents and the

OSTE base material. In contrast, surface functionalization via TEC of Vim and Aam hc could

be clearly confirmed by a significant increase in nitrogen (N1s) content, from 4.5 (OSTE) to

7.3 and 5.5 atom%, respectively. Vim is a reactive monomer and known to polymerize under

radical condition, which could explain the relatively strong increase in nitrogen content using

Vim.47 Consequently, the high reactivity of Vim could have led to the formation of short

polymer grafts on the surface, even at low concentrations and short reaction times.

In addition to XPS analysis, the impact of the different surface modifications was also

investigated by water contact angle (WCA) measurements. The WCA of APFB reacted

surface (62.4°) changed slightly compared to the reference surface (OSTE, 67.0°), which was

assumed to be the result of the low reactivity, as already seen from the XPS data. A similar

trend was observed for OSTE-ATFA, where the WCA was reduced more significantly to

41.1° upon modification. This was attributed to the polar nature of ATFA. As expected,

increased hydrophobicity of the surface was achieved via functionalization with OFPB, which

was confirmed by a WCA of 77.0°. Reduced WCAs of 35.0° (OSTE-AA), 25.1° (OSTE-

AMA) and 55.2° (OSTE-AGE) were observed due to the introduction of hydroxyl, carboxylic

acid and epoxide groups via surface TEC with AMA, AA and AGE. Similarly, WCAs of

38.5° (OSTE-Vim) and 55.4° (OSTE-Aam hc) validated the functionalization with Vim and

Aam hc. This illustrates the versatility of the system providing successful surface

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modification by TEC and that the surface properties of the microplate wells could be adjusted

by the formation of a functional monolayer.

Additionally, thiol groups are known to serve as chain transfer agents in free radical

polymerization reactions in order to reduce molecular weight of the polymers. Surface bound

thiols can act in a similar way as reported earlier for other types of surfaces, where the surface

could be grafted by termination of growing polymer chains.48–50 In this study, we have

expanded this SCT-FRP approach as an alternative method to TEC for controlled surface

modification of OSTE materials. Different acrylic and methacrylic monomers containing

polyethylene glycol (PEG) (via MPEGMA), a zwitterionic sulfobetaine (SBMA), fluorine

(OFPA) and epoxide groups (GMA) were utilized. In this case, by running the photochemical

reaction at a higher concentration, polymer grafting onto the well surfaces could be achieved

by SCT-FRP in the presence of DMPA as radical photoinitiator, as shown in Scheme 1.

Typically, the liquid monomers were used in a 1:2 volume ratio in ethanol, whereas SBMA,

being a solid, was applied in a concentration of 2.4 M. In contrast to TEC, surface

modification via SCT-FRP can be confirmed by IR spectroscopy, as illustrated in Figure 2.

Figure 2 IR spectra of virgin OSTE surface and OSTE grafted via SCT FRP with various

acrylate and methacrylate based polymer, such as MPEGMA, GMA, SBMA or OFPA

4000 3500 3000 2500 2000 1500 1000 500

2572

OSTE-g-pOFPA

OSTE-g-pSBMA

OSTE-g-pGMA

OSTE-g-pMPEGMA764

1141145916771729

603

wavenumber (cm-1)

2963

2872

2936

OSTE

852

757904989

841

17331684

1461 1035

1101

944

764

1129

14491685

1724

525763

10361143

1176

145916761727

3439 2965

2975

539765806

17361684

1463

1165 1128

901

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A clear indication of the formation of a thick surface layer by SCT-FRP is the full

disappearance of the S-H stretch band at 2572 cm-1 upon surface polymerization of each

monomer. The spectrum of OSTE-g-pMPEGMA showed a strong absorption at 1100 cm-1,

which can be assigned to C-O-C stretch vibration originating from the PEG side chain. An

additional band at 904 cm-1 for the OSTE-g-pGMA is observed, which is the epoxide ring

vibration. In the IR spectrum of the OSTE surface upon grafting with pSBMA, a band at 3439

cm-1 indicates the ammonium N-H stretch vibration. Furthermore a broadening of the C-O-C

stretch absorption at 1143 cm-1 as well as the strong band at 1036 cm-1 confirms the presence

of pSBMA. Surface grafting with pOFPA led to the appearance of carbon-fluorine bands at

1163, 806 and 539 cm-1. XPS and WCA analysis corroborated these results, which are

presented in Table 2.

Table 2. XPS data and static water contact angles (WCA) of virgin and grafted OSTE surfaces via SCT FRP with a variety of acrylate and methacrylate based polymersa.

C1s O1s N1s S2p F1s WCA

[atom%] [atom%] [atom%] [atom%] [atom%] [°]

OSTE 61.1

(60.6) 23.3±0.2

(24.9) 4.5±0.3

(4.1) 11.1±0.3

(10.4) 67.0±1.2

OSTE-g-pMPEGMA

65.6±0.6 (67.6)

30.1±0.2 (32.4)

0.5±0.4 (0.0)

3.9±0.2 (0.0)

25.4±3.6

OSTE-g-pGMA

70.7±0.4 (70.0)

28.7±0.4 (30.0)

0.5±0.1 (0.0)

69.6±7.5

OSTE-g-pSBMA

62.9±0.0 (61.1)

25.2±0.0 (27.8)

4.7±0.1 (5.6)

7.2±0.1 (5.6)

20.4±4.8

OSTE-g-pOFPA

43.2±0.6 (44.4)

11.1±0.3 (11.1)

0.5±0.1 (0.0)

1.3±0.3 (0.0)

44.0±1.3 (44.4)

118.1±1.9

a) In parenthesis, theoretical atom composition of the virgin surface (OSTE) and monomers

used for SCT FRP, results are based on three replicated measurements on the same surface

(n=3)

XPS analysis of a pMPEGMA grafted surface (OSTE-g-pMPEGMA) showed significantly

increased carbon and oxygen contents, which is consistent with the theoretical value of

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MPEGMA. Combination with the simultaneous decrease in sulfur and nitrogen confirms the

polymer grafting of the OSTE-g-pMPEGMA surface. Similar XPS results were achieved by

polymer grafting with pGMA, where the oxygen and carbon content increased approaching

the theoretical value of the monomer. The amount of nitrogen and sulfur was even lower

compared to OSTE-g-pMPEGMA, indicating an even thicker pGMA layer. The theoretical

atom composition of SBMA is more similar to that of the OSTE background, which leads to

only minor changes in the atom composition as a result of grafting. However, the content of

the individual atoms from the OSTE-g-pSBMA surface approximated the theoretical values of

the SBMA monomer, substantiating the success of the grafting reaction. Compared to the

aforementioned surface modification via TEC with OFPA, polymerization conditions (higher

concentration, longer reaction time) lead here to the appearance of a much higher fluorine

content of 44.0 atom% compared to the 7.1 atom% by TEC. Additionally, the total atom

composition of the OSTE-g-pOFPA surface was found to be in good agreement with that of

the pure monomer, which confirms the formation of a polymer layer on the surface under

SCT-FRP conditions.

These presented changes in atom composition, upon polymer grafting with the individual

monomers, were corroborated by the variation in WCAs of the reacted surfaces. Grafting with

pMPEGMA increased the hydrophilic character, which was shown by a reduction in WCA

from 67° (OSTE) to 25.4°. Similarly, a substantial hydrophilization was achieved by grafting

with pSBMA (20.4°) confirming the surface reaction. The WCA increased slightly upon SCT-

FRP using GMA (69.6°). OSTE-g-pOFPA exhibits a very high WCA of 118.1° and

demonstrates the drastically increased hydrophobicity of the surface upon polymer grafting,

which is significantly higher than the one obtained from TEC using the same monomer

(77.0°). These results, together with the earlier discussed XPS and IR data, illustrate the

potential for altering surface properties through selection of reaction conditions. Low

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concentrations and short reaction times lead to addition of the acrylate to the thiol groups

based on TEC. Increased concentrations and longer reaction times favor polymerization,

which undergoes chain transfer or termination with surface thiols leading to a thicker surface

coating. The application of either surface modification method, TEC or SCT-FRP,

demonstrates the versatility of these grafting reactions in order to achieve high control over

the surface functionality and properties.

The functionalized well bottom surfaces were subsequently used for enzyme immobilization.

For this purpose, a rhodamine labelled horseradish peroxidase (Rho-HRP) dissolved in PBS

buffer (pH7.3) was used as a model enzyme. Fluorescence intensity (FI) measurements were

used to confirm the presence of immobilized enzyme on all surfaces. In order to evaluate

qualitatively the amount of immobilized enzyme, FI measurements (λexcitation = 544 nm,

λemission = 595 nm) of the surfaces were conducted before (as reference) and after incubation

with Rho-HRP. Figure 3 shows the reference corrected FI measurements of enzyme exposed

TEC (A) and SCT-FRP modified surfaces (B).

Figure 3. Reference corrected fluorescence intensity of virgin (OSTE) and functionalized

surfaces via TEC (A) and SCT FRP (B) after immobilization of rhodamine labeled

horseradish peroxidase (Rho-HRP), excitation at 544 nm and emission at 595 nm, standard

deviations are based on three experimental replicates (n=3)

OSTEOSTE-

APFB

OSTE-

ATFA

OSTE-

OFPA

OSTE-

AA

OSTE-

AMA

OSTE-

Vim

OSTE-

Aam hc

OSTE-

AGE

0

20

40

60

80

100

A

corrected fluorescence intensity (ex544/em

595)

OSTE OSTE-g-

pMPEGMA

OSTE-g-

pSBMA

OSTE-g-

pOFPA

OSTE-g-

pGMA

0

20

40

60

80

100

B

corrected fluorescence intensity (ex544/em

595)

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It can be seen that the native OSTE surface exhibited a substantial FI upon exposure to Rho-

HRP (Figure 3A, OSTE), which relates to significant loading of labeled enzyme on the

surface. TEC functionalized surfaces, such as APFB, ATFA, AA, AMA and Vim provided

similar FI results and consequently comparable adsorption of Rho-HRP. Higher FI results are

the consequence of OPFA modifications, which is an indication for increased enzyme loading.

Accordingly the lower FI values of Aam hc compared to other functionalized surfaces suggest

a lower enzyme coverage. Compared to all aforementioned surfaces, surface functionalization

with epoxide groups due to the reaction with AGE leads to a drastic increase in FI upon

exposure to enzyme, which is more than 6-fold higher compared to that of the native OSTE

surface. Amine groups from lysine or thiols from cysteine residues within the enzyme

structure are expected to react covalently with epoxide groups on the surface and thus create a

higher enzyme loading.51

Similar measurements were performed with surfaces functionalized by SCT-FRP, as shown in

Figure 3B. The highest FI was observed for pMPEGMA modified surfaces. This result was

unexpected, since PEG surface grafts were reported in the literature to exhibit anti-fouling

properties and therefore reduced unspecific protein adsorption.52,53 In order to explain this

discrepancy, FI of Rho-HRP was measured in solution in the presence of different MPEGMA

concentrations, as presented in Figure S5. These results show a direct correlation of FI with

increasing amounts of MPEGMA. This effect of FI enhancement for Rho-HRP in the

presence of MPEGMA could indicate an artificially high loading of enzyme on the OSTE-g-

pMPEGMA surfaces. Good biocompatibility and anti-fouling properties have also been

described for zwitterionic polymers, such as pSBMA.54–56 Herein, pSBMA grafted surfaces

with an increased hydrophilicity, show a very low FI after enzyme immobilization, which

reinforces the hypothesis of low enzyme loading on these surfaces. Surface functionalization

by pOFPA under SCT-FRP conditions shows slightly increased FI compared to the native

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OSTE surface, which is similar to that of TEC functionalized surface with OFPA (Figure 3A).

Relatively similar enzyme loading based on comparable FI results were achieved by surface

grafting with pGMA via SCT-FRP, even though the epoxide containing pGMA enables

covalent immobilization.

A significant advantage of this platform is that biocatalytic activity of immobilized enzymes

could be measured spectrophotometrically directly in a microplate reader by using a

colorimetric assay. For immobilized Rho-HRP on the previously prepared surfaces by either

TEC or SCT-FRP, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt

(ABTS) was used as a colorimetric assay. The slope of absorbance, which correlated directly

to the formed product, over time, was used to express the initial enzymatic activity of the

particular surfaces, as shown in Figure 4A for TEC modified surfaces.

Figure 4. Initial enzyme activity of Rho-HRP immobilized on virgin (OSTE) and

functionalized surfaces via TEC (A) with various ene compounds and SCT FRP (B) with

various acrylate and methacrylate based polymers, standard deviations are based on three

experimental replicates (n=3)

From these results, a clear difference in Rho-HRP activity from the individual surface

modification, upon enzyme immobilization, can be seen. In general, the displayed activities

are caused by two factors, enzyme loading and biocatalytic activity, which both directly

OSTEOSTE-

APFB

OSTE-

ATFA

OSTE-

OFPA

OSTE-

AA

OSTE-

AMA

OSTE-

Vim

OSTE-

Aam hc

OSTE-

AGE

0,00

0,02

0,04

0,06

0,08

0,10

0,12

initial activity (∆abs m

in-1)

A

OSTE OSTE-g-

pMPEGMA

OSTE-g-

pSBMA

OSTE-g-

pOFPA

OSTE-g-

pGMA

0,00

0,02

0,04

0,06

0,08

0,10

0,12

B

initial activity (∆abs m

in-1)

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affected the overall activity. This directly reflects the influence of the individual surface

chemistry. The native OSTE surface exhibited an activity of 0.022 ∆abs min-1. Even though

TEC modified surfaces with fluorinated (APFB, ATFA and OFPA), hydroxyl (AA) and

carboxylic acid containing compounds (AMA) expressed a substantial enzyme loading, these

were not active at all (see Figure 4A), which suggested an unfavorable environment for the

enzyme by these functional groups. On the contrary, imidazole (Vim) and amine (Aam hc)

functional surfaces showed the highest initial activities for TEC modified surfaces of about

0.023 and 0.018 ∆abs min-1, respectively. These results indicate that amine, imidazole as well

as thiol groups from the native OSTE surface provide a more beneficial local environment

towards the enzyme and thus, activity was retained. FI indicated high enzyme coverage on

surfaces, functionalized with epoxide groups (OSTE-AGE). However, the resulting initial

activity was only 0.009 ∆abs min-1, which is significantly lower than that of OSTE, Vim and

Aam hc surfaces. It was assumed that this is a result of unfavorable interaction of the surface

with the enzyme, which is known to have a significant impact during the adsorption-covalent

immobilization mechanism onto epoxy supports.6 As a consequence, blocking of the active

site or conformational changes of the enzyme could have resulted in the low activity.

Likewise, the initial enzyme activity was determined from surfaces grafted with polymer

layers by SCT-FRP after Rho-HRP immobilization (see Figure 4B). Hydrophilic surfaces due

to grafting with pMPEGMA and pSBMA tend to be enzymatically inactive. The low activity

of OSTE-g- pSBMA correlates directly with the low enzyme loading, which was determined

by FI measurements. The low activity of pMPEGMA grafted OSTE surfaces relates well with

the anti-fouling nature of PEG grafted surfaces. Their tendency to FI enhancement indicates

an artificially high enzyme loading. Surfaces which were grafted with hydrophobic pOFPA by

SCT-FRP, exhibit a similar activity (0.019 ∆abs min-1) compared to the original OSTE

surface, which correlates well with the results from the FI measurements. The activity was

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found to be substantially higher than those of surfaces with OFPA monolayer

functionalization via TEC, even though both surfaces show comparable enzyme loadings.

Thus, increased hydrophobic interactions between the enzyme and the surface created by a

thicker surface layer seems to have a positive effect on the enzyme activity A high HRP

activity (0.07 ∆abs min-1) can be seen from OSTE-g-pGMA surfaces prepared by SCT-FRP.

GMA based polymers, bearing epoxide groups, allow covalent attachment of HRP and has

already been used in various studies for enzyme immobilization.57,58 Compared to the epoxide

functional monolayer formed from AGE by TEC, the pGMA surface layer shows a decreased

enzyme loading, but substantial improvement of enzymatic activity.

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4. Conclusions

A single, versatile platform for testing a broad variety of surface chemistries as candidates for

supports for enzyme immobilization is proposed in this study, with the main objective of

making identification of suitable surface/enzyme combinations in a more facile and time

saving manner. This strategy indeed permits a faster, easier and broader surface–enzyme

screening compared to the traditional “trial and error” method generally involving resins. The

results showed that the STE/OSTE microplate is suitable for colorimetric measurements

above 340 nm and the thiol functional wells can be functionalized through either TEC or

SCT-FRP providing a broad selection of functional surfaces. We have shown how TEC/SCT-

FRP can be exploited to prepare functional monolayers (TEC) or thicker polymer layers

(SCT-FRP). Thus, different surface functionalities, such as hydroxyl, carboxylic acid, amine,

fluorine, imidazole, epoxide, PEG and zwitterionic groups could be introduced, which was

confirmed by XPS analysis. Through immobilization of HRP as a model enzyme, the effects

of surface/enzyme interactions were illustrated in the microplate, showing clear correlations

between surface functionalities and enzymatic activities. HRP displayed improved activities

when attached directly to imidazole, thiol and amine functional surfaces, compared to

hydroxyl, fluorinated, carboxylic acid or epoxide containing surfaces. Immobilization of HRP

on pOFPA modified surfaces demonstrated a significant activity, which might be caused by

increased hydrophobic interactions between enzyme and surface. Based on the initial

biocatalytic activities relative to the surface chemistry it is possible to identify candidates that

should be tested in depth for enzyme immobilization. Thereby we have demonstrated the

potential of this screening platform to be used for other enzymes, facilitating the identification

of suitable surfaces for immobilization. Furthermore, by use of such a platform it would be

possible to determine the influence of other parameters, such as temperature, pH, and e.g.

substrate concentration.

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Supporting Information

Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements

The authors wish to thank the Aage and Johanne Louis-Hansens Endowment for financial

support.

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